Clathrin-coated vesicles (CCVs) mediate endocytosis of receptor- bound nutrients, hormones and proteins destined for degradation, as well as receptor sorting in the trans-Golgi network required for biogenesis of lysosomes and secretory granules. Self- assembly of the triskelion-shaped clathrin molecule into a polyhedral protein coat is the driving force for receptor sequestration by CCVs. The mammalian clathrin triskelion comprises three heavy chains and three light chains of two different types, LCa and LCb, with splicing variants. The light chain subunits regulate clathrin assembly and disassembly, and receptors become associated with the polymerized clathrin lattice via adaptor molecules, which also regulate clathrin self- assembly. This proposal aims to define the molecular basis for clathrin self-assembly by resolution of the crystallographic structure of clathrin, as a foundation for structure-based mutagenesis. During the first funding period of this grant, we crystallized and solved the structure of the proximal domain of the clathrin triskelion. This revealed a structural motif, termed clathrin heavy chain repeat (CHCR), which we predict, by sequence profile, may account for clathrin heavy chain trimerization interactions. The CHCRs are repeated throughout the linear portion of the clathrin triskelion leg, terminating at the flexible linker region attached to the terminal domain, whose structures were recently solved by the groups of Kirchhausen and Harrison. The CHCR was also found in other proteins involved in membrane traffic to the yeast vacuole. We propose to complete the structural determination of the remainder of the clathrin triskelion leg (the trimerization domain, the distal leg segment and several forms of the clathrin light chain subunits bound to the clathrin heavy chain) and will test the general function of the CHCR structural motif. This analysis of clathrin light chains will help elucidate how they exert regulation and contribute to understanding their differential function. As the structures of the clathrin segments are determined, they will be fit into a 21 angstrom resolution model of assembled clathrin, produced by Pearse and colleagues, to determine how clathrin segments interact in the polyhedral lattice. Crystal forms of self-assembled sub-fragments will also be analyzed to establish assembly interactions. Lastly, the models of subunit and assembly interactions will be tested by structure-based mutagenesis. Understanding the molecular control of clathrin polymerization involved in formation of CCVs is relevant to cell growth control, to maintenance of homeostasis by receptor and ligand downregulation, and to membrane traffic pathways generating an immune response.